Hedgehog信号通路在咀嚼力缺乏牙槽骨中的表达变化

doi:10.3969/j.issn.1005-4979.2022.01.002

摘要/Abstract

摘要：

目的：观察发育过程中咀嚼力缺乏对小鼠下颌牙槽骨改建的影响，并探讨Hedgehog信号通路在该过程中的表达变化。方法：选取3周龄C57BL/6J雄性小鼠，将其随机分为2组，分别通过正常饮食（hard diet, HD）和软食（soft diet, SD）喂养1周和3周, 取小鼠下颌骨，应用micro-CT扫描及组织学染色法[HE染色、Goldner三色染色、抗酒石酸酸性磷酸酶（TRAP）染色、免疫荧光染色]对骨改建情况进行分析。选取下颌磨牙区牙槽骨并提取RNA，行实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction, RT-qPCR），研究Hedgehog信号通路在骨改建中的变化情况。结果：喂养1周后，软食组小鼠根分叉区牙槽骨量即出现显著降低；喂养3周后，其骨量丢失进一步加重。组织学染色结果发现，软食组根方1/2区域牙槽骨矿化程度显著降低，矿化区域显著减少，且破骨活动显著增强。Hedgehog信号通路下游分子Smoothened（SMO）、Patched1（PTCH1）、神经胶质瘤相关癌基因同源蛋白1（glioma-associated oncogene homolog 1, GLI1）表达上调。结论：下颌骨发育过程中，咀嚼刺激不足表现为牙槽骨量降低，Hedgehog通路相关分子SMO、PTCH1、GLI1、IFT88表达均上调，提示Hedgehog信号通路在该过程中起重要作用。

关键词: Hedgehog信号通路, 咀嚼力, 骨改建, 软食

Abstract:

Objective: The purpose of this study was to observe the effect of masticatory force deficiency on the remodeling of mandibular alveolar bone in mice, and to explore the expression of Hedgehog signaling pathway in this process. Methods: 3-week-old male C57BL/6J mice were selected and randomly divided into two groups. They were treated with hard diet (HD) or soft diet (SD) respectively for 1 week or 3 weeks. After the mandibles were removed, the micro-CT scan and histological stainings (HE, Goldner trichrome, TRAP and immunofluorescence) were used to analyze bone remodeling. RNA was extracted from the alveolar bone in the mandibular molar area, and real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect the changes of the Hedgehog signaling pathway during bone remodeling. Results: After feeding soft food for 1 week, the bone mass in the root bifurcation area decreased significantly. After 3 weeks, the level of bone loss further increased. By histological staining, the degree of mineralization and mineralized area of the SD group were significantly reduced too, and the osteoclast activity was obviously enhanced, mainly in the alveolar bone near the apical 1/2 area. The downstream molecules of Hedgehog signaling pathway, including Smoothened (SMO)、Patched1 (PTCH1) and glioma-associated oncogene homolog 1(GLI1) were up-regulated. Conclusion: During the development of mandible, insufficient chewing stimulation results in decreased alveolar bone volume. The expression levels of SMO, PTCH1, GLI1 and IFT88 in the Hedgehog pathway were up-regulated, suggesting that the Hedgehog pathway plays an important role in this process.

Key words: Hedgehog signaling pathway, masticatory force, bone remodeling, soft diet

张帅, 扈梓悦, 牛萍萍, 孙瑶. Hedgehog信号通路在咀嚼力缺乏牙槽骨中的表达变化[J]. 《口腔颌面外科杂志》, 2022, 32(1): 7-13.

ZHANG Shuai, HU Ziyue, NIU Pingping, SUN Yao. Expression of Hedgehog signaling pathway in alveolar bone with masticatory deficiency[J]. 《Journal of Oral and Maxillofacial Surgery》, 2022, 32(1): 7-13.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2022/V32/I1/7

图/表 6

表1 引物序列

Table 1 Primer sequences

基因	引物序列5′-3′	引物序列3′-5′
GAPDH	TGTGTCCGTCGTGGATCTGA	CCTGCTTCACCACCTTCTTGA
IFT88	TGAGGACGACCTTTACTCTGG	GAAAACCCGTGTCATTCTCCAA
SMO	GGGCTGGGAGTCGGTTTTAAT	CTTCAACCCTGGGAACCCTC
PTCH1	TGCTATGACTGCCTGGTTGG	AGCCCAACACCTGGTTCAAA
GLI1	ACTCTGGGATATGGGGGACC	CAGGGCCATAGTTGGTTGGT

图1 Micro-CT扫描和定量分析结果 A—C. 4周龄正常饮食组小鼠下颌骨及第一磨牙ROI的CT扫描图；D—F. 4周龄软食组小鼠下颌骨及第一磨牙ROI的CT扫描图；G—J. 4周龄软食组及正常饮食组ROI骨质BV/TV、Tb.N、Tb.Th、Tb.Sp定量分析；K—M. 6周龄正常饮食组小鼠颌骨及第一磨牙ROI的CT扫描图；N—P. 6周龄软食组小鼠下颌骨及第一磨牙ROI的CT扫描图；Q—T. 6龄软食组及正常饮食组ROI骨质BV/TV、Tb.N、Tb.Th、Tb.Sp定量分析。*表示P<0.05，**表示P<0.01，与正常饮食组相比。

Figure 1 Micro-CT scans and quantitative analysis results

图2 小鼠下颌第一磨牙HE染色结果 A、E、I、M. 2组4周龄和6周龄小鼠HE染色图，虚线框内为ROI（×40）；B、F、J、N. 2组 4周龄和6周龄小鼠ROI的HE 染色图，虚线将其分为冠方与根方区域（×100）；C、G、K、O. 2组4周龄和6周龄小鼠根分叉冠方区域的HE染色图（×200）；D、H、L、P. 2组4周龄和6周龄小鼠根分叉根方区域的HE染色图（×200）。

Figure 2 HE staining results of mouse mandibular M1

图3 第一磨牙牙槽骨DMP1及Goldner三色染色结果 A、D、K、N. 2组 4周龄和6周龄小鼠第一磨牙根分叉区域DMP1免疫荧光染色图（×100）；B、E、L、O. 2组4周龄和6周龄小鼠根分叉冠方区域DMP1免疫荧光染色图（×200）；C、F、M、P. 2组4周龄和6周龄小鼠根分叉根方区域DMP1免疫荧光染色图（×200）；G、I、Q、S. 2组4周龄和6周龄小鼠根分叉冠方区域Goldner三色染色图（×200）；H、J、R、T. 2组4周龄和6周龄小鼠根分叉根方区域Goldner三色染色图（×200）。

Figure 3 DMP1 and Goldner trichrome staining results of alveolar bone of M1

图4 M1牙槽骨TRAP及CTSK染色结果 A、D、K、N. 2组 4周龄和6周龄小鼠第一磨牙根分叉区TRAP染色图（×100）；B、E、L、O. 2组 4周龄和6周龄小鼠根分叉冠方区域TRAP染色图（×200）；C、F、M、P. 2组 4周龄和6周龄小鼠根分叉根方区域TRAP染色图（×200）；G、I、Q、S. 2组 4周龄和6周龄小鼠根分叉冠方区域CTSK免疫荧光染色图（×200）；H、J、R、T. 2组 4周龄和6周龄小鼠根分叉根方区域CTSK免疫荧光染色图（×200）。

Figure 4 TRAP and CTSK staining results of alveolar bone of M1

图5 Hedgehog通路及初级纤毛相关基因表达情况 A. 2组4周龄和6周龄小鼠SMO的mRNA表达水平；B. 2组4周龄和6周龄小鼠PTCH1的mRNA表达水平；C. 2组4周龄和6周龄小鼠GLI1的mRNA表达水平；D. 2组4周龄和6周龄小鼠IFT88的mRNA表达水平。**表示P<0.01，***表示P<0.001，与正常饮食组相比。

Figure 5 The expression of Hedgehog pathway and primary cilia related genes

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